The present invention relates to improved methods and apparatus for mass spectrometry. In particular the invention provides methods and apparatus that dissociate ions in an ion reservoir prior to mass spectrometric analysis. The methods and apparatus of the invention can be used in the analysis of ions of peptides, proteins, carbohydrates, oligonucleotides, nucleic acids, and small molecules as prepared by combinatorial or medicinal chemistry.
Mass spectrometry (MS) is a powerful analytical tool for the study of molecular structure and interaction between small and large molecules. The current state-of-the-art in MS is such that less than femtomole quantities of material can be readily analyzed using mass spectrometry to afford information about the molecular contents of the sample. An accurate assessment of the molecular weight of the material may be quickly obtained, irrespective of whether the sample""s molecular weight is several hundred, or in excess of a hundred thousand, atomic mass units or Daltons (Da). Mass spectrometry can elucidate significant analytical aspects of important biological molecules. One reason for the utility of MS as an analytical tool is the availability of a variety of different MS methods, instruments, and techniques which can provide different pieces of information about the samples.
A mass spectrometer analyzes charged molecular ions and fragment ions from a sample molecule. These ions and fragment ions are then sorted based on their mass to charge ratio (m/z). A mass spectrum is produced from the abundance of these ions and fragment ions that is characteristic of every compound. In the field of biotechnology, mass spectrometry can be used to determine the structure of a biomolecule. Of particular interest is the ability of mass spectrometry to be used in determining the sequence of oligonucleotides, peptides, and oligosaccharides.
Various mass spectrometric techniques can be used to deduce the sequence of an oligonucleotide. Murray, K. K., J Mass Spec., 1996, 31, 1203-1215, which is incorporated herein by reference in its entirety. Two commonly used ionization methods are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Mass spectrometry is also commonly used for the sequencing of peptides and proteins (see, in general, Biemann, K., Annu. Rev. Biochem., 1992, 61,977-1010, which is incorporated herein by reference in its entirety).
In principle, mass spectrometers consist of at least four parts, (1) an inlet system, (2) an ion source, (3) a mass analyzer and (4) a mass detector/ion-collection system. Skoog, D. A. and West, D. M., Principles of Instrumental Analysis, Saunders College, Philadelphia, Pa., 1980, 477-485. The inlet system permits the sample to be introduced into the ion source. Within the ion source, molecules of the sample are converted into gaseous ions. The most common methods for ionization are electron impact (EI), electrospray ionization, chemical ionization and matrix-assisted laser desorption/ionization (MALDI). A mass analyzer resolves the ions based on mass-to-charge ratios. Analyzers can be based on magnetic means (sector), time-of-flight, quadrupole and Fourier transform mass spectrometry (FTMS). A mass detector collects the ions as they pass through the detector and records the signal. Each ion source can potentially be combined with each type of mass analyzer generating a wide variety of mass spectrometers.
The field of mass spectrometry is rapidly evolving. Improvements in mass spectrometric instrumentation and methodologies are needed to address increasingly challenging applications in a number of research arenas including the physical, biological, and medical sciences. In many implementations ofmass spectrometers based on Penning and Paul traps, ion formation, isolation, and detection take place in the same region of a vacuum chamber and are temporally, rather than spatially, separated. In a typical pulse, sequence ions are alternatively formed and detected; the ionization duty cycle is defined as the fraction of time ions are formed compared to the overall experiment time. Thus, in high resolution measurements, which may take several seconds to perform yet require ionization intervals of only a few milliseconds, the overall ionization duty cycle is only a few percent. A number of approaches have been explored to improve the ionization duty cycle including schemes in which ions are formed and continuously accumulated in an external ion reservoir and periodically gated into the mass analyzer. For example, a Penning trap in the fringing magnetic field of an Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was used to accumulate ions formed by EI during high resolution measurements in the FTICR cell. Hofstadler, S. A. and Laude, D. A, Jr., Anal. Chem., 1991, 63, 2001-2007, which is incorporated herein by reference in its entirety. Senko, M. W. et al. (J. Amer. Soc. Mass Spectrom., 1997, 8, 970-976) demonstrated that an external ion reservoir formed by an rf-only multipole bounded by two electrostatic elements can efficiently accumulate ions generated by electrospray ionization and the ion ensemble can be periodically pulsed into the FTICR cell for mass analysis.
Another means of improving mass spectra is the use of dissociation to fragment the molecular ions. Dissociation strategies for tandem ESI-MS can be separated into two general categories: those which take place in the ESI source prior to mass analysis, and those which take place after the ESI source and often rely on some form of m/z dependent ion manipulation. For example, Loo, J. A. et al. (Anal. Chim. Acta, 1990, 241, 167-173) demonstrated that large multiply charged proteins could be effectively dissociated by employing a relatively large voltage difference between the exit of the desolvating capillary and the skimmer cone. Similarly, Rockwood, A. L. et al. (Rapid Comm. Mass Spectrom., 1991, 5, 582-585) demonstrated that ions could be thermally dissociated in the ESI source by heating the desolvation capillary to extreme temperatures. Both of these xe2x80x9cin-sourcexe2x80x9d dissociation schemes produce mass spectra which are rich in fragment ions and can provide sequence information for peptides, proteins, or oligonucleotides. Alternatively, a number of post-source dissociation schemes have been presented which are now widely employed. In general, scanning MS/MS instruments such as triple quadrupoles and magnetic sector instruments employ collisionally activated dissociation (CAD) to effect the dissociation of an m/z selected parent ion. Dagostino, P. A., et al., J Chrom., 1997, 767, 77-85. In addition to employing various forms of CAD (Gauthier, J. W., etal., Chim. Acta, 1991, 246, 211-225; and Senko, M. W., et al., Anal. Chem., 1994, 66, 2801-2808), FTICR instruments have successfully demonstrated the use of UV-photodissociation (Williams, E. R., et al., J. Amer. Soc. Mass Spectrom., 1990, 1, 288-294), infrared multiphoton dissociation (IRMPD) (Little, D. P., et al., Anal. Chem., 1994, 66, 2809-2815), surface induced dissociation (SID) (Ijames, C. F. and Wilkins, C.L ., Anal. Chem., 1990, 62, 1295-1299; and Williams, E. R., et al., J Amer. Soc. Mass Spectrom., 1990, 1, 413-416), blackbody infrared radiative dissociation (BIRD) (Price, W. D., et al., Anal. Chem., 1996, 68, 859-866), and more recently, electron capture dissociation (ECD) (Zubarev, R. A., et al., J Am. Chem. Soc., 1998, 120, 3265-3266) to fragment precursor ions.
Infrared multi-photon dissociation (IRMPD) uses photodissociation generally in combination with FTICR or quadrupole ion trap mass analyzers. In this method, ions are collected in the FTICR analyzer cell and the laser interacts with ions within the cell. In IRMPD, the laser dissociates ions into fragment ions, as opposed to an ionization method involving lasers, e.g. MALDI. The most common method of ionization used in IRMPD methods is electrospray ionization as this provides more highly charged ions that are more easily dissociated, as compared to MALDI. Little, D. P., et al., Anal. Chem., 1994, 66, 2809-2815. Little, D. P., et al. used IRMPD for protein and nucleotide sequencing. IRMPD has also been used with quadrupole ion trap mass spectrometers. Colorado, A., et al., Anal. Chem., 1996, 68, 4033-4043.
Currently, IRMPD methods are limited to mass spectrometers based on FTICR and QIT. With FTICR methods the kinetic energy release which accompanies the dissociation event can cause a redistribution of the ions in the trapped ion cell. Upon excitation, these ions can obtain a range of cyclotron radii, which precludes high performance mass measurements. Also, the laser irradiation interval is identical for each ion, which limits the dissociation pathways available to the ion.
Although improvements have been made in the mass spectrometric analysis of biomolecules, especially with the use of IRMPD, there remains a need for improved mass spectrometric methods and apparatuses.